Proteomic Analysis of Redox-Dependent Changes Using Cysteine-Labeling 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 117

5. Bromophenol blue: 0.05% (w/v) bromophenol blue in H 2 O. To
make 10 mL, weigh 5 mg bromophenol blue and make to
10 mL with distilled H 2 O. Filter and store at room temperature.

2.3. Preparation
of 2D-Gels, Imaging,
and Image Analysis

1. Bind-Silane solution: For twelve 24 × 20 cm plates, mix 12 mL
of PlusOne Bind-Silane (GE Healthcare), 300 mL glacial acetic
acid, 12 mL ethanol and 2.7 mL ddH 2 O.
2. PlusOne Repel-Silane solution (GE Healthcare).
3. Immobiline DryStrip pH 3-10 NL gel strips (GE Healthcare).
4. Mineral oil.
5. Equilibration buffer: 6 M urea, 30% (v/v) glycerol, 50 mM
Tris-HCl pH 6.8, 2% (w/v) SDS. To make 200 mL, dissolve
72 g urea in 100 mL distilled H 2 O. Add 60 mL of 100% glyc-
erol, 10 mL of 1 M Tris pH 6.8 solution, and 4 g SDS. Dissolve
all powders and adjust volume to 200 mL with ddH 2 O. Aliquot
and store at −20°C.
6. Ammonium persulfate (APS): Prepare a 10% solution in ddH 2 O
and store at 4°C for no more than a month.
7. Gel preparation: 30% acrylamide/bisacrylamide solution,
1.5 M Tris-HCl, pH 8.8, 10% SDS, 10% APS, N,N,N,N ¢-
tetramethylethylenediamine (TEMED).
8. Agarose overlay: 0.5% (w/v) low-melting point agarose in
SDS-PAGE running buffer. To make 200 mL, melt 1 g of
agarose in 200 mL of 1× SDS-PAGE running buffer in a
microwave on low heat. Add bromophenol blue solution to
give a pale blue color.
9. SDS-PAGE running buffer (10×): Tris-glycine SDS buffer
10× (Severn Biotech LTD). Store at room temperature.
10. Ettan DALT twelve Large Vertical Electrophoresis System (GE
Healthcare).
11. Typhoon 9400 Variable Mode Imager and ImageQuant
software (GE Healthcare).
12. DeCyder software (v7.0) (GE Healthcare).

1. Colloidal CBB fi xing solution: 35% (v/v) ethanol, 2% (v/v)
phosphoric acid in ddH 2 O.
2. Colloidal CBB staining solution: 34% (v/v) methanol, 17%
(w/v) ammonium sulfate, and 3% (v/v) phosphoric acid in
ddH 2 O (see Note 4).
3. Coomassie Blue G-250.
4. Bio-Rad GS-800 scanning densitometer and QuantityOne
software (Bio-Rad Laboratories Inc. USA.) or Typhoon 9400
Variable Mode Imager and ImageQuant software (GE
Healthcare).
5. Ettan Spot Picker (GE Healthcare).

2.4. Post-Staining
and Spot Excision